In utero hematopoietic cell transplantation (IUHCT) is a promising therapeutic approach potentially applicable to a large number of genetic diseases. Utilizing the murine model of IUHCT, we have established mixed hematopoietic chimerism across full MHC barriers and gained understanding into the requirements for engraftment and associated donor specific tolerance. We have determined the mechanism of tolerance after IUHCT, and utilized prenatal tolerance to facilitate postnatal bone marrow transplantation and immunotherapy with conversion of partial to complete donor chimerism across full MHC barriers without toxicity. Our long-term objective is the translation of these successful strategies to clinical application, and the development of novel and safe new approaches to achieve therapeutic engraftment by IUHCT alone. In this proposal our specific aims are: 1) To define the components and mechanism of the adaptive immune response that determine the success or failure of allogeneic engraftment after IUHCT. We have observed that there are clear differences in the frequency of engraftment between congenic and allogeneic IUHCT using BM and HSC. These differences have been unmasked by our current technique of intravascular injection of large doses of cells. In this aim we will investigate the hypothesis that many donor reactive cells escape clonal deletion, and that the maintenance of chimerism is dependent upon generation of an adequate peripheral regulatory response prior to rejection of donor cells. 2) To enhance engraftment by specifically targeted prenatal immunotherapy. We will investigate the use of minor histocompatibility immunodominant antigens expressed predominantly on hematopoietic cells as a target for specific immunotherapy to enhance engraftment in the fetus without GVHD. 3) To investigate selective strategies to improve homing and engraftment of donor cells. We have also identified a mechanism to improve homing and engraftment of donor cells after in utero transplantation by blockade of the dipeptidylpeptidase CD26. The studies in this proposal are designed to develop and optimize novel approaches to IUHCT that will be safely translatable to large animal and human systems. The ultimate goal of these studies is to develop safe and effective strategies that can be translated to the in utero treatment of a broad range of human genetic hematologic disorders including Sickle Cell Disease and Thalassemia.